Biosynthesis of palladium, platinum, and their bimetallic nanoparticles using rosemary and ginseng herbal plants: evaluation of anticancer activity

In this research, palladium (II) and platinum (II), as well as their bimetallic nanoparticles were synthesized using medicinal plants in an eco-friendly manner. Rosemary and Ginseng extracts were chosen due to their promising anticancer potential. The synthesized nanoparticles underwent characterization through FT-IR spectroscopy, DLS, XRD, EDX, SEM, and TEM techniques. Once the expected structures were confirmed, the performance of these nanoparticles, which exhibited an optimal size, was evaluated as potential anticancer agents through in vitro method on colon cancer cell lines (Ls180, SW480). MTT assay studies showed that the synthesized nanoparticles induced cell death. Moreover, real-time PCR was employed to investigate autophagy markers and the effect of nanoparticles on the apoptosis process, demonstrating a significant effect of the synthesized compounds in this regard.


Anticancer
In recent times, substantial research efforts have been dedicated to the synthesis of various metal nanoparticles, including but not limited to silver, palladium, platinum, gold, iron, zinc using environmentally friendly methods.These nanoparticles have been explored for their catalytic and biological applications [1][2][3][4] .Over the past decade, there has been a noticeable shift towards green synthesis methods, involving the identification and optimization of these methods, along with investigating the structure and characteristics of synthesized nanoparticles [5][6][7] .In more recent studies, particular emphasis has been placed on the practical applications of synthesized nanoparticles across various domains [8][9][10][11][12] .For example, nanoparticles prepared using different microorganisms, including bacteria, algae, yeast, and fungi, and extracts of various plant components (leaf, root, stem, fruit) have shown various applications in both catalysis and bio-medical field 13,14 .Researches in recent years show that metal and metal oxide nanoparticles prepared using green methods have shown important potential environmental and biomedical applications [15][16][17][18][19][20] .Among various metallic nanoparticles, the synthesis of platinum and palladiumbased nanoparticles via various methods has been at the center of attention, primarily owing to their outstanding usages the field of medicine.Throughout history, platinum-based medications, notably cisplatin and carboplatin, have been employed in cancer chemotherapy.More recently, platinum nanoparticles (PtNPs) have emerged as a subject of interest.Additionally, palladium nanoparticles (PdNPs) have demonstrated potential for combating leukemia and breast cancer.While various physical and chemical synthesis methods exist for these nanoparticles, biological approaches are favored for their superior biocompatibility and non-toxic nature, which is essential for effective nanomedicine [21][22][23][24] .Palladium and platinum nanoparticles, synthesized through diverse methods have exhibited notable antimicrobial and anticancer properties [25][26][27] .Numerous studies have proven their role in inducing apoptosis in cancer cells [28][29][30] .Additionally, the therapeutic potential of medicinal plant extracts in cancer treatment has been the subject of extensive investigation [31][32][33][34] .So far, considerable research has been conducted in the field of biological investigation and pharmacological effects of medicinal plant extracts, with particular emphasis on the plants employed in this study, namely rosemary and ginseng.Several reports indicate

Preparation of rosemary and ginseng extracts
To create rosemary plant extract, in the first step, the process began by thoroughly washing the needle-shaped leaves with distilled water and allowing them to air-dry at room temperature.Once completely dried, were fragmented into smaller pieces, and a fine, consistent powder was produced through grinding.In the following, 10 g of this plant powder were mixed with 100 mL of deionized water and heated to boiling (100 °C) over a 15-min duration.Then, the reaction container was left to gradually cool.The resulting solution was subsequently filtered through the use of Whatman No. 1 filter paper and maintained at 4 °C, ready for utilization in the subsequent phases of the experiments.
The procedure for preparing the red ginseng root extract closely resembled the previously described method.To begin, 25 g of ginseng root were rinsed a couple of times by distilled water and dried.Following this, it was divided into smaller pieces and completely smoothed and uniformed using a mortar.Subsequently, the ginseng mixture was subjected to boiling in 100 mL of deionized water for a duration of 30 min.After the cooling phase, similar to the previous method, the plant extract was filtered using Whatman filter paper, and the resulting solution underwent centrifugation at 10,000 rpm for 10 min to separate other suspended compounds from the primary extract.The resulting solution was then diluted to 100 mL using deionized water and stored at 4 °C as a stock solution for future experimentation.

Synthesis of palladium nanoparticles
To achieve this, the process began with the preparation of a 10 mM aqueous solution of PdCl 2 .Then, 5 mL of rosemary extract was introduced into 95 mL of the metal ion solution.Following that, 100 µL of concentrated hydrochloride were added to the solution to convert PdCl 2 into H 2 PdCl 4 •nH 2 O.Then, 1.11 g of PVP was dissolved in methanol and incorporated into the reaction mixture to enhance solubility and stability in aqueous medium.A 20 mL of methanolic NaOH solution was introduced drop by drop into the reaction mixture, and the solution was refluxed for 5 h at 100 °C.At the end, the resulting solution was put in the refrigerator, and after the desired period, sedimentation and solution stability were assessed.It was determined that the use of PVP yielded homogenous nanoparticles to an acceptable level, with no precipitation observed even after six months.Synthesis of palladium nanoparticles using ginseng extract was also done according to the same method except for the extract type.

Synthesis of platinum nanoparticles
A 10 mM aqueous solution was prepared using H 2 PtCl 6 •6H 2 O. Subsequently, 5 mL of rosemary extract was inserted into 95 mL of metal solution.Following this step, 1.11 g of PVP were dissolved in methanol and added to the reaction mixture to enhance solubility and stability in aqueous medium.The reaction mixture is refluxed for 5 h at 100 °C.Finally, the resulting solution was refrigerated and after the specified timeframe, the level of sedimentation and solution stability were assessed.It was observed that the use of PVP yielded homogeneous nanoparticles to a satisfactory extent.The synthesis of platinum nanoparticles using ginseng extract followed the same procedure, differing only in the extended reaction time, which lasted for 6 h.
In all reactions, the determination of reaction completion was based on referenced articles and the observation of a shift in color from yellow to brownish-black

Synthesis of bimetallic nanoparticles
To this end, different ratios (1:1, 1:2, and 2:1) of palladium and platinum metal ions were investigated.Following the method employed for monometallic nanoparticle synthesis, a 10 mM solution containing both metals was prepared.First, the palladium solution was introduced into the reaction container, and 100 µL of concentrated hydrochloride was added.Subsequently, the platinum solution was incorporated, followed by stirring vigorously.In this phase, 5 mL of plant extract was inserted, followed by the addition of 1.11 g of PVP, which was dissolved before introducing 20 mL of methanolic NaOH solution drop by drop into the reaction medium.The reaction mixture was refluxed for 3 h at 100 °C.All these steps were repeated for Pd/Pt concentration ratio of 1:2 and 2:1.Three reactions were performed using rosemary extract and three reactions were performed using ginseng extract.Finally, six blackish-brown solutions were obtained.

Cell lines, culture, and treatment
Human colorectal cancer cell lines, specifically SW480 and LS180, were purchased from Bonyakhteh Company located in Tehran, Iran.We conducted experiments using cells within passages 3-6, obtaining fresh and validated cell lines from the same source after 6 passages.These cells were nurtured in Dulbecco's Modified Eagle's Medium high in glucose and glutamine (DMEM) provided by Bio Idea in Tehran, Iran.The culture medium was further enriched with 10% Fetal Bovine Serum (FBS) (Gibco™; Cat #: 16000044) and 1% penicillin-streptomycin (Gibco, (Waltman, MA, USA).They were stored in a humidified incubator with an atmosphere consisting of 95% air and 5% CO 2 , all at a temperature of 37 °C.

MTT assay
The practicability of cells under varying empirical conditions was assessed using the MTT assay, which quantified the growth inhibitory impact of the compounds on cancer cell lines.This was achieved through the application of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 78 .A known concentration of tumor cells introduced into the wells of a 96-well plate, and various compound concentrations (ranging from 0 to 1000 µmol dm −3 ) were applied.The plate was then incubated at 37 °C with 5% CO 2 and 95% humidity for a duration of 24-48 h.Subsequently, 10 µL of MTT solution (5 mg/mL) was introduced into each well, thereafter followed by an additional 4-h incubation at 37 °C.The formazan produced, which was insoluble, was eventually dissolved by the addition of dimethyl sulfoxide (DMSO) at a volume of 100 µL per well.Following the complete dissolution of the dye, we measured the optical density (OD) at 570 nm using a reference wavelength of 630 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek's ELx808, USA).To determine the percentage of cell inhibition resulting from various treatments, we used the following formula: % Inhibition = 100 − [(test OD/non-treated OD) × 100].It's important to note that in all experiments, the non-treated cultures consisted solely of DMSO solvent at a concentration equivalent to that in the test wells.We constructed an inhibition percentage graph across different concentrations to establish the values for the half-maximal inhibitory concentration (IC 50 ) 79,80 .

RNA extraction and real-time PCR (RT-PCR) analysis
The quantitative reverse-transcriptase polymerase chain reaction (q RT-PCR) technique was exploited to assess the relative expression levels of Beclin-1, LC3, and P62.Total RNA was extracted from sub-confluent cell cultures, in line with the manufacturer's guidelines.Subsequently, cDNA synthesis was carried out using the cDNA synthesis Kit from Yekta Tajhiz, Iran.Primer sequences (as detailed in Table 1) were designed with the assistance of AlleleID software (version 7.73).All samples were scrutinized in duplicate, and a no-template control (NTC) was included using sterile nuclease-free water (ddH 2 O).The Q RT-PCR was executed using the QuantStudio™ 3 Real-Time PCR System (ABI Applied Biosystems) and SYBR Green Amplicon from Yekta Tajhiz, Iran.The relative expression levels of the target mRNAs were standardized to the internal standard, GAPDH expression levels.In the end, the relative quantification was conducted using the Applied QuantStudio™ Design and Analysis Software.

Synthesis and characterization of nanoparticles
The freshly prepared nanoparticles solutions underwent immediate dynamic light scattering (DLS) analysis.
The resulting nanoparticle sizes can be found in Table 2.The findings revealed that employing various concentrations of metal salt solution in the synthesis reaction led to the formation of nanoparticles of varying sizes.
Considering that the concentration of the metal solution affects the reaction speed, it also affects the size of the produced nanoparticles.Of course, other factors such as the nature of metal ions, the nature and concentration of the plant solution, the ratio of plant solution to metal solution, pH, temperature, and reaction duration, Table 1.The primer sequences employed in this study.

Gene Forward primer Reverse primer
Beclin www.nature.com/scientificreports/exerted an influence on nanoparticle shape and size 81 .For the synthesis of monometallic nanoparticles, the same concentration of metal solution was used to investigate the effect of the nature of the metal ions and the plant extracts.On the other hand, during the synthesis of bimetallic nanoparticles by changing the ratio of metals, it was observed that the nature of the metal has a significant effect on the size of the nanoparticles.The particle size analysis confirmed that all particles are synthesized in the nanometer scale.Smaller particles were formed in slower reactions, indicating that the rate of nanoparticle formation directly impacted their size.For example, platinum reactions, which were slower than palladium reactions, led to the synthesis of generally smaller nanoparticles.Also, in bimetallic nanoparticles, an increased ratio of palladium to platinum resulted in larger particles.The reason why platinum reactions are slower than palladium reactions can be explained by accounting the position of these two elements in the periodic table.On the other hand, reactions involving rosemary extract occurred at a faster rate than those using ginseng extract, leading to the production of larger nanoparticles.Note that the compounds existing in the plant extract influenced both the mechanism and the speed of the reaction 69 .
Furthermore, a small amount of colloidal solutions from each sample was applied to a silicon substrate for the purpose of conducting XRD characterizations.The structure of the nanoparticles was examined in grazing incidence mode (with θ inc = 0.1 Â°) using a rotating anode of Cu Kα radiation (λ = 1.540598Å) operating at 45 kV  To conduct scanning electron microscopy (SEM), the nanoparticles were dispersed in water, and a drop of this suspension was deposited onto a carbon specimen sample holder.Figure 4 presents the SEM images of nanoparticles.In Fig. 4B, cubic nanoparticles with an average diameter of 94.12 ± 26.47 nm are shown, while Fig. 4D shows that the majority of particles have a spherical shape with an average particle size of 139.22 ± 53.99 nm.It is worth noting that the observed sizes are larger than those obtained through XRD patterns.The XRD sizes are attributed to crystalline sizes, while the SEM images may depict the size of several agglomerated crystals.Furthermore, the relevant EDX elemental mapping (Fig. 5) demonstrated a well-distributed composition of elements in the synthesized nanoparticles.For nanoparticle 7, the atomic percentages were as follows: 86.915, 10.995, 0.695, and 1.395 for C, O, Pd, and Pt, respectively.Correspondingly, for nanoparticle 10, the atomic percentages were 85.005, 13.485, 0.535, and 0.975, respectively.The results affirm the presence of the extract compounds in the nanoparticles compositions, thus corroborating the findings of the FT-IR analysis.
To further investigate the morphology of the nanoparticles, a droplet of the synthesized colloidal solutions for each sample was deposited onto a TEM grid and allowed to air-dry. Figure 6A is a TEM image of 7 NPs.This image reveals that the majority of particles exhibit diameters measuring approximately 3.7 ± 0.8 nm, although smaller and larger NPs are also present.The 10 NPs appear to be predominantly spherical, consistent with the TEM image depicted in Fig. 6.The distribution of diameters indicates a mean value of approximately 2.3 (± 0.1) nm.

In vitro selective cancer cell cytotoxic activity
The cytotoxic activity of the nanoparticles against SW480 and LS180 colorectal cancer cell lines was assessed through the MTT dye reduction method, a well-established bioassay used to measure the extent of an agent's specific detrimental effect on certain cells.Cytotoxicity, indicating the toxicity level towards cells, was evaluated by incubating cells with increasing nanoparticle concentrations for 24-48 h, followed by assessing anti-proliferative activity (see "Materials and methods" section).Table 3 and Fig. 7 provide the cytotoxicity data, represented as IC 50 values, which correspond to the concentrations of a compound inhibiting 50% of cell growth.The results highlight that the degree of lethality and cytotoxicity of the synthesized nanoparticles are influenced by the type of plant extract employed in their synthesis.Moreover as can be seen in Fig. 7, different concentrations of nanoparticles solution were treated on the cell lines and compared with the control.The results showed that the presence of nanoparticles in the culture medium prevented cell survival and growth, and compared to the control culture medium that did not receive any drug, the cytotoxicity rate increased in the treatment culture mediums with increasing concentration.Many studies have investigated the effect of metal nanoparticles, especially palladium and platinum nanoparticles, on colon cancer cell lines.For example, in a research, it was determined that the effective dose of a bimetallic palladium-platinum nanoparticle on the SW480 cell line is 50 µg/mL 83 .In addition, in another study, the effect of 75 µg/mL of palladium-platinum bimetallic nanoparticles on the same cell line has been reported 84 .It should be noted that the concentration values reported in these studies are lower than the values obtained in our research, but the current research is acceptable to a large extent due to the fact that an environmentally friendly synthesis method was used for the synthesis of nanoparticles.It seems that the method of synthesis and the used solvent have a great effect on the purity and anti-cancer property of the  nanoparticle.Furthermore the effective doses indicate that the achieved concentrations are notably more efficient than those reported in previous studies involving plant extracts alone on colorectal cell lines.The results of rosemary extract on SW480 cell line have shown that the IC 50 value is 500 µg/mL 48 .Consequently, it is evident that nanoparticles outperform the plant extracts 48,85 .
Research on the function and mechanism of different antitumor drugs such as 5 fluorouracil (5-FU), mitomycin (MMC), cisplatin (CP), all-trans retinoic acid (ATRA), TRAIL, doxorubicin (DOX) and oxaliplatin (OP) has been done on the SW480 cell line [86][87][88][89] .For example, the IC 50 values reported for oxaliplatin and DOX on this cell line are 2.1 ± 0.3 and 65.25 ± 3.48 µM, respectively 88,90 .Also, the effect of known antitumor drugs on LS180 cell line has been investigated 91 .For instance, the IC 50 values for cisplatin, oxaliplatin and carboplatin on LS180 cell lines are 4.1 ± 0.1, 1.7 ± 0.8 and 147.1 ± 19.4 μM, respectively 92 .Although these drugs have excellent efficacy, due to the side effects and drug resistance they cause, scientists have recently paid attention and focus to the use of drugs with less damage.

Nanoparticles effect on the expression levels of autophagy-related genes
Researchers have illuminated a set of mechanisms in the toxicity of nanoparticles such as apoptosis, necrosis, oxidative stress and autophagy.Among the mechanisms, autophagy was recently recognized as an important style of cell death in various toxicity caused by nanoparticles.Macroautophagy consists of three steps: cytoplasmic constituents (mis-folded proteins or damaged organelles) are enveloped by double-membraned autophagosome; autophagosomes are fused with lysosome to form an autolysosome; and the cytoplasmic constituents are degraded and recycled in lysosome.Autophagy is likened to the opposite faces of Janus, as it could protect cells to survive under certain severe conditions, as well as inducing cell death when too much autophagy occurs.Similarly, nanoparticles-mediated autophagy during nanotoxicity might be an adaptive cellular response aiding in the clearance of nanoparticles, and also might be harmful in cellular dysfunction 93 .
Research conducted in recent years indicate that various metal and metal oxide-based nanoparticles can show good anti-cancer properties by affecting the autophagy process 94 .For example, silver nanoparticles reduced the viability of PANC-1 pancreatic adenocarcinoma cells and induced apoptotic and autophagic cell death significantly more than non-tumor cells.In addition, the level of autophagy marker protein LC3-II was significantly increased in PANC-1 cells treated with silver nanoparticles, thus indicating that apoptotic and necroptosis cell death occurs with autophagy 95 .In recent research, it was found that silver nanoparticles affect the function of lysosome and autophagic flux by reducing the expression of TFEB in A459 lung cancer cells and lead to cell damage 96 .In another research, it was found that gold nanoparticles cause autophagy at the same time as oxidative stress on human lung cancer cell line.Also, the formation of autophagosomes along with the absorption of nanoparticles in lung fibroblasts and the positive regulation of autophagy proteins, microtubule-associated protein 1 light chain 3 (MAP-LC3) and autophagy gene 7 (ATG7) were observed in the treated samples 97 .Some researchers have developed various forms of autophagy-targeted nanodrug delivery systems to treat tumors, leading to advances in cancer therapy.The mechanism of autophagy induction by nanomaterials is thought to be mainly mediated by intracellular oxidative stress 98 .
The studies revealed that both nanoparticles have a significant impact on the expression of autophagy genes, with varying results based on the specific gene, the duration and dosage of the nanoparticles, and the nature of the cell line.As shown in Fig. 8, when examining the effect of nanoparticle 7 on the LS180 cell line, it becomes apparent that the Beclin-1 gene experiences an increase after 24 h at concentrations of 250 and 500 µg/mL.This suggests the initiation of the autophagy process, and after 48 h, autophagy is inhibited at all concentrations.So it can be said that this process may be dependent on time and dosage, having a prolonged impact on cancer cells On the other hand, the LC3 gene shows an increase only at the concentration of 1000 µg/mL.This observation indicates autophagosome accumulation within 24 h, followed by autophagy flux inhibition at higher concentrations, eventually leading to cellular toxicity and apoptosis.In other words, lower concentrations induce autophagy initiation, while higher concentrations result in toxicity.Over the 48-h period, autophagy inhibition occurs initially.Consequently, if we aim for a rapid impact, lower concentrations are preferable, while for longer-term applications, the difference in concentration is less crucial due to early autophagy inhibition.The behavior of the P62 gene resembles that of LC3, with the most pronounced effect observed at the 1000 µg/mL concentration after 24 h, followed by the early-stage inhibition of autophagy after 48 h.
Regarding nanoparticle 10, akin to nanoparticle 7, there is an increase in autophagy activity within the initial 24 h.In a shorter timeframe, the autophagy flux is inhibited, eventually leading to the final stage of autophagy being blocked.Conversely, over a more extended period, the initial phase of autophagy is inhibited.Overall, in all experiments, autophagy flux inhibition was consistently observed at the highest concentration within the initial 24-h interval (see Fig. 8).
When it comes to the SW480 cell line, the results are different.Examining the effect of nanoparticle 7 on Beclin-1 and LC3 genes shows a decrease in gene expression in a shorter timeframe, followed by an increase over a more extended period.Notably, the P62 gene demonstrated more pronounced effects at 48 h compared to 24 h, with a general lack of dose dependence.To elaborate, the initial stage of autophagy was impacted at 24 h, while the autophagy flux was affected at 48 h (see Fig. 9).Nanoparticle 10 was also investigated on the SW480 cell line.Beclin-1 results showed that it inhibited autophagy at low concentrations, but stimulated the initial stages of autophagy at a concentration of 300 µg/mL, similarly occurring over 48 h.As for LC3 and P62 genes, low concentrations of nanoparticles within 24 h hindered the initial stage of autophagy, with the 150 µg/mL concentration proving to be notably effective.But in 48 h, both concentrations inhibited the autophagy flux (see Fig. 9).The SW480 cell line has c-myc, Kras, and p53 oncogenes.Kras and c-myc mutation lead to autophagy activity, and P53, a key player in autophagy, is compromised in this cell line.Considering the influence of these oncogenes on autophagy, it is highly likely that they disrupt the normal and logical processes in this cell line.

Conclusions
Present paper employed an environment-friendly approach to synthesize palladium and platinum nanoparticles.For this purpose, the synthesis utilized extracts from rosemary and ginseng plants, both known for their anticancer properties.Then, the size and structure of these nanoparticles were scrutinized through the use of conventional methods, like DLS analysis, FT-IR spectroscopy, XRD, SEM, EDX and TEM techniques, revealing their nanoscale dimensions suitable for biological applications.Two bimetallic nanoparticles (7 and 10NPs) were chosen for further investigation and evaluation with cell cultures.The findings indicate a significant impact of both nanoparticles on the tested cancer cell lines.The results showed that the nanoparticles synthesized by an environmentally friendly method with rosemary and ginseng extract had an acceptable effect on colon cancer cell lines (SW480 and LS180), which had a better effect compared to the plant extract alone.Furthermore, the study explored the influence of nanoparticles on the expression of autophagy-related genes, yielding diverse outcomes based on nanoparticle concentration, experiment duration, gene type, and cell line characteristics.

Figure 2 .
Figure 2. FT-IR spectra of Rosemary extract and 7 NPs (A), Ginseng extract and 10 NPs (B).In both pictures the upper spectrum is related to plant extract and the lower spectrum is NPs.

Table 2 .
DLS size of the synthesized nanoparticles.It should be noted that Rz, Gn, Pd, and Pt are abbreviations of Rosemary, Ginseng, Palladium, and Platinum, respectively.Also, the numbers indicate the concentration ratio of metal solutions during synthesis.Significant values are in bold.

Table 3 .
Selective cytotoxicity data, stated as IC 50 , of the compounds opposing cancer cells.